Method of inducing labor or treating pre-eclampsia by administering relaxin analogs

ABSTRACT

Human relaxin analogs, polypeptide compositions related thereto, as well as nucleotide compositions encoding the same, are provided.

GOVERNMENT RIGHTS

The invention was made with government support under contract HD047606awarded by the National Institutes of Health. The Government has certainrights in the invention.

The present invention relates to relaxin analogs, and uses thereof. Thepresent invention further relates to compositions and formulationscomprising relaxin analogs and their uses. The present invention alsorelates to compositions and formulations comprising the relaxin analogs,its derivatives and/or relaxin or other agent wherein such compositionexhibits an additive or synergistic effect.

BACKGROUND OF THE INVENTION

Relaxin is a pregnancy hormone discovered in 1926 (Hisaw (1926) Proc.Soc. Exp. Biol. Med. 23: 661-663), based on its ability to relax thepublic ligament in guinea pig. Mature human relaxin is a hormonalpeptide of approximately 6000 daltons known to be responsible forremodelling the reproductive tract before parturition, thus facilitatingthe birth process. A concise review of relaxin was provided by Sherwood,D. in The Physiology of Reproduction, Chapter 16, “Relaxin”, Knobil, E.and Neill, J., et al. (eds.), (Raven Press Ltd., New York), pp. 585-673(1988). Relaxin has local autocrine and/or paracrine roles thatcontribute to connective tissue remodeling at the maternal-fetalinterface during late pregnancy and at parturition, including anincrease in the expression of the genes, proteins, and enzyme activitiesof the matrix metalloproteinases interstitial collagenase (MMP-1),stromelysin (MMP-3), and gelatinase B (MMP-9).

In humans, the relaxin gene family contains a total of seven members:relaxin H1 (RLN1), relaxin H2 (RLN2), relaxin 3/INSL7 (RLN3), INSL3/RLF,INSL4/EPIL, INSL5/RIF2, and INSL6/RIF1 (Hudson et al. (1983) Nature301:628-631; Hudson et al. (1984) EMBO Journal 3:2333-2339; U.S. Pat.Nos. 4,758,516 and 4,871,670). The primary translation product of H2relaxin is a preprorelaxin consisting of a 24 amino acid signal sequencefollowed by a B chain of about 29 amino acids, a connecting peptide of104-107 amino acids, and an A chain of about 24 amino acids. Among thesefamily members, RLN2 and INSL3 signal through two leucine-richrepeat-containing GPCRs, LGR7 (RFXR1) and/or LGR8 (RFXR2). Whereas RLN2is capable of activating both LGR7 and LGR8, INSL3 is a selective ligandfor LGR8. In addition to the better characterized RLN2 and INSL3, RLN3was shown to activate LGR7, GPCR135, and GPCR142, but not LGR8 (6-10);therefore, the relaxin family peptides exhibit overlapping specificityon the activation of LGR7 and LGR8.

Evidence has accumulated to suggest that relaxin is more than a hormoneof pregnancy and acts on cells and tissues other than those of thefemale reproductive system. Relaxin causes a widening of blood vessels(vasodilatation) in the kidney, mesocaecum, lung and peripheralvasculature, which leads to increased blood flow or perfusion rates inthese tissues (Bani et al (1997) Gen. Pharmacol. 28, 13-22). It alsostimulates an increase in heart rate and coronary blood flow, andincreases both glomerular filtration rate and renal plasma flow (Bani etal (1997) Gen. Pharmacol. 28, 13-22). The brain is another target tissuefor relaxin where the peptide has been shown to bind to receptors. Inaddition to the role in remodeling of reproductive tissues, relaxin hasbeen shown to effect endometrial differentiation during embryoimplantation, nipple and mammary gland development, angiogenesis, woundhealing, and renal cardiovascular responses. Furthermore, recent studieshave shown that relaxin prolongs the survival of tumor-bearing mice byenhancing the degradation of the extracellular matrix, thereby slowingdown tumor growth.

In contrast, INSL3 is essential for testis descent in rodents andcontributes to the regulation of, 1) male germ cell apoptosis; 2)initiation of meiotic progression of arrested oocytes in preovulatoryfollicles; and 3) the positioning of the female gonad duringdevelopment. Although the importance of LGR7 and LGR8 in human RLN1 andRLN2 signaling remains to be studied, in vitro evidence indicated thathuman RLN2 could effect LGR8 signaling in vivo. Based on its pleiotropiceffects on tissue remodeling, relaxin has been the subject for clinicaltrials aimed to treat scleroderma, pre-eclampsia, congestive heartfailure, and to enhance cervical ripening during the third trimester ofpregnancy. The finding that human RLN2 is capable of activating bothLGR7 and LGR8 raises the possibility that clinical applications of humanRLN2 could pose unwanted responses in the LGR8 signaling pathway inpatients, which could include effects on spermatogenesis and ovarianfollicle development.

Therefore, a better understanding of the molecular mechanisms underlyingthe interaction of human RLN2 and its receptors, as well as thegeneration of an LGR7-specific human relaxin analog are of greatinterest.

SUMMARY OF THE INVENTION

Relaxin analogs, polypeptide compositions related thereto, as well asnucleotide compositions encoding the same, are provided. The analogpolypeptides of the invention comprise at least one amino acidsubstitution relative to the wild-type protein, and have alteredreceptor selectivity or activity relative to the wild-type protein. Thepeptides may be provided as pharmaceutically acceptable compositions forhuman or animal administration, by various therapeutic routes. Peptidesmay be isolated in purified or homogenous form free of contaminatingpeptides and proteins, or in a form of about 90-99% purity.

The relaxin analogs of the invention have altered receptor specificityas compared to the reference, naturally occurring forms, e.g. wild-typehuman RLN2. In some embodiments, a Type I analog is provided, which hasa reduced affinity for the receptor LGR8 (RFXR2); and/or an increasedaffinity for the receptor LGR7 (RFXR1). In other embodiments, a Type IIanalog is provided, which exhibits an enhanced bioactivity upon bothreceptors. Polypeptides of interest include processed forms of therelaxin analogs of the invention, which comprise an A chain and a Bchain, which A chain and B chain may be linked through a disulfide bond;and unprocessed forms in which the A chain and B chain are linkedthrough the C chain or through a truncated C chain. In otherembodiments, an A chain analog is provided.

The analogs are useful as therapeutic agents. Conditions treatable withrelaxin analogs include, without limitation, conditions that benefitfrom collagen or extracellular matrix remodelling. Included areinduction of labor, treatment of pre-eclampsia, congestive heartfailure, treatment of endometriosis, treatment of skin conditions suchas scleroderma, treatment of fibrosis, treatment of hypertension.Relaxin has been implicated in the dilation of blood vessels' smoothmuscle cells, for treatment of hypertension. Relaxin is also been usefulin the treatment of severe chronic pain, particularly pain arising fromstretching, swelling, or dislocation of tissues.

Human wild-type RLN2 is capable of activating both LGR7 and LGR8, andtherefore clinical applications of human RLN2 may pose unwantedresponses in the LGR8 signaling pathway in patients, which could includeeffects on spermatogenesis and ovarian follicle development. The type Irelaxin analogs of the invention allow relaxin treatment to specificallytarget the LGR7 receptor, which is the prime target for most clinicalapplications of relaxins. Type II analogs, which have increased overallpotencies on the two relaxin receptors relative to the wild-typepolypeptide, find use clinically where receptor selectivity is notrequired.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Syntenic mapping of the RLN1/RLN2/INSL4/INSL6 locus intetrapods. Schematic representation of relaxin locus syntenic to theRLN1/RLN2/INSL4/INSL6 locus on human chromosome 9 in chimpanzee (P.troglodytes), Rhesus monkey (M. mulatta), cow (B. taurus), dog (C.familiaris), mouse (M. musculus), rat (R. norvegicus), the grayshort-tailed opossum (M. domestica), platypus (O. anatinus), chicken (G.gallus), and the clawed frog (X. tropicalis). The relaxin family geneson the syntenic relaxin family locus B (RFLB) locus of differentvertebrates are indicated by black rectangles, whereas, neighboringgenes are indicated by blank rectangles. The chromosomal numbers or thegenomic contig numbers are indicated at the bottom of the schematicrepresentation of each genomic fragment. The positions of select genesin the genomes of human and X. tropicalis are also indicated (*), partof the syntenic region on chromosome 8 of cow is mapped to unknownchromosome in the GenBank™.

FIG. 2. Alignment of amino acid sequences of the B and A chains ofrelaxin peptides from the RFLB of 17 representative tetrapods as setforth in SEQ ID NO:7-24. Residues that are completely conserved in allspecies analyzed are highlighted by a dark background. Resides that areconserved in at least five sequences are indicated by a gray background.Positions of residues are indicated by numbers on top of the alignment.

FIG. 3. Generation and characterization of tagged recombinant human RLN2peptide. A, schematic design of recombinant RLN2 peptides with anN-terminal Myc tag and a His₆ tag at four different positions of themature peptides (RLN2 His1-4). The mature B and A chains are indicatedby a rectangle box, whereas the C domain is indicated by a line. Myc andHis₆ epitopes are indicated by a light blue rectangle and a yellowrectangle, respectively. B, Western blotting analysis of tagged RLN2peptides in conditioned media of transfected HEK293T cells using ananti-relaxin antibody (Calbiochem; upper panel) or an anti-Myc antibody(lower panel) under reducing conditions. The molecular weight standardis shown on the left. C, stimulation of cAMP production by RLN2 His1 andRLN2 His2 peptides in LGR7-expressing HEK293T cells.

FIG. 4. Characterization of recombinant human RLN2 peptides generatedusing expression constructs with a truncated C domain linker sequence.A, schematic design of RLN2 expression constructs with a truncated Cdomain (RLN2 C-8 to C-38). The linker sequence was derived from thefirst 38-amino acids of the human RLN2 C domain and was flanked by apair of dibasic cleavage sites for convertase cleavage. B, Westernblotting analysis of a nontagged RLN2 peptide and the tagged RLN2peptide generated using the expression construct with an 8-amino acid Cdomain sequence. Specific bands are detected using an anti-relaxinantibody, and are indicated by arrows. The molecular weight standard isshown on the left. C, stimulation of cAMP production by RLN2 peptides inLGR7-expressing HEK293T cells. Each data point represents the mean±S.E.of quadruplicate samples. D, competitive LGR7-binding analysis of theMyc- and His₆-tagged RLN2 C-8. Each data point represents the mean±S.E.of triplicate samples.

FIG. 5. Stimulation of cAMP production in LGR7- and LGR8-expressingcells by recombinant RLN2, RLN3, and INSL3. A, recombinant RLN3 andINSL3 selectively activated LGR7 and LGR8, respectively. In contrast,RLN2 activates both receptors at the nanomolar range. Unlike the wildtype RLN2, substitution of Arg^(B12) and Arg^(B16) residues with alanineabolished the LGR7- and LGR8-activation activities of RLN2. Each datapoint represents the mean±S.E. of quadruplicate samples. B, Westernblotting analysis of recombinant wild type and mutant relaxin peptidesincluding, RLN2 C-104, C-38, C-28, C-18, C-8 (upper left), WT RLN2, WTRLN3, WT INSL3 (upper middle), WT RLN2, RLN2 R^(B12)A, RLN2 R^(B16)A(upper right), WTRLN2, RLN2 T^(A16)A, RLN2^(A19-20), RLN2^(A22-23)(lower left), WT RLN2, RLN2 K^(A17)A, RLN2 R^(A18)A, RLN2 R^(A22)A, RLN2F^(A23)A (lower middle), WT RLN2, RLN2 K^(A17)A, RLN2 K^(A17)G, RLN2K^(A17)Q, and RLN2 K^(A17)D (lower right). Affinity column purifiedpeptides (100 ng/lane) were resolved by 18% SDS-PAGE under nonreducing(upper panel) and reducing (lower panel) conditions. The positions ofmolecular mass markers are indicated on the left.

FIG. 6. Alanine substitution in the C terminus of A chain alters thereceptor-activation activity of RLN2. A, schematic representation ofRLN2 mutants with alanine substitution at five different positions ofthe A chain (RLN2 T^(A16)A, RLN2 K^(A17)A, RLN2 R^(A18)A, RLN2^(A19-20),and RLN2^(A22-23)) shown in SEQ ID NO:25-26. B, stimulation of cAMPproduction in LGR7- and LGR8-expressing cells by the wild type andmutant RLN2 peptides. Unlike the wild type peptide, RLN2 T^(A16)A andRLN2 K^(A17)A mutants exhibited a significantly enhanced LGR8-activationactivity. C, competitive LGR7- and LGR8-binding assays of wild type andmutant RLN2 peptides. Each data point in the receptoractivation andreceptor-binding assays represents the mean±S.E. of quadruplicate andtriplicate samples, respectively.

FIG. 7. Alanine substitution at the Phe^(A23) position alters thereceptor-interaction activities of RLN2. A, schematic representation ofRLN2 mutants with alanine substitution at the Arg^(A22) or Phe^(A23)positions shown in SEQ ID NO:27-28. B, stimulation of cAMP production inLGR7- and LGR⁸-expressing cells by RLN2 R^(A22)A and RLN2 F^(A23)Amutants. F^(A23)A mutation ablated LGR⁸-activation activity. C, LGR⁷-and LGR⁸-binding activities of R^(A22)A and F^(A23)A mutant peptides.F^(A23)A mutation significantly reduced LGR⁸-binding activity, butwithout an effect on LGR7-binding activity. Each data point in thereceptor-activation and receptor-binding assays represents the mean±S.E.of quadruplicate and triplicate samples, respectively.

FIG. 8. Comparative structure modeling of mutant peptides based on thecrystal structure of human RLN2. Three-dimensional structure of the wildtype human RLN2 and the K^(A17)A mutant based on the RLN2 crystalstructure (PDB 6RLN). The wild type RLN2 structure is shown on the leftand right panels. The K^(A17)A mutant is shown on the middle panel. TheA and B chains are indicted by white filled space and green dot space,respectively. The amino acids at A16, A17, and A23 positions areindicated by the brown filled space. The Arg^(B12), Arg^(B16), andIle^(B19) residues in the RXXXRXXI motif of B chain are represented bythe blue filled space.

FIG. 9. Stimulation of cAMP production in LGR7- and LGR8-expressingcells by mutant peptides with residue substitution at the Lys^(A17)position. A, stimulation of cAMP production in LGR7- and LGR8-expressingcells by K^(A17)A, K^(A17)G, K^(A17)D, and K^(A17)Q mutants. Unlike theK^(A17)A peptide, mutants with an aspartic acid, glycine, or glutamineat the Lys^(A17) position did not exhibit an enhanced LGR8-activationactivity. Each data point represents the mean±S.E. of quadruplicatesamples. B, LGR7- and LGR8-binding activities of K^(A17)A, K^(A17)G,K^(A17)D, and K^(A17)Q mutants. Each data point represents the mean±S.E.of triplicate samples.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Relaxin analogs, polypeptide compositions related thereto, as well asnucleotide compositions encoding the same, are provided. The subjectpolypeptide and/or nucleic acid compositions find use in a variety ofdifferent applications, including in vivo prophylactic and therapeuticpurposes; as immunogens for producing antibodies; in screening forbiologically active agents; and the like.

It is shown herein that an amino acid substitution at one or more of SEQID NO:3 residue 16, and SEQ ID NO:3 residue 17, where the naturallyoccurring amino acids threonine and lysine are replaced with a smallneutral amino acid results in enhanced LGR8-activation activity. In someembodiments of the invention, a relaxin analog comprises an amino acidsubstitution at one or both of SEQ ID NO:3 residue 16, and SEQ ID NO:3residue 17, wherein the analog has increased bioactivity relative to thewild-type polypeptide.

It is also shown herein that that an amino acid substitution at SEQ IDNO:3 residue 23, where the naturally occurring amino acid phenylalanineis replaced with a small neutral amino acid ablates LGR8, but not LGR7,activation activity. In some embodiments of the invention, a relaxinanalog comprises an amino acid substitution at SEQ ID NO:3 residue 23,wherein the analog has a reduced affinity for the receptor LGR8 relativeto the wild-type polypeptide.

Relaxin is a hormone with a number of important functions, which includethe modulation of the reproductive physiology of human beings and othermammals, including, but not limited to, maintaining pregnancy, effectingparturition, and enhancing sperm motility as an aid in fertilization.Relaxin has significant effects on connective tissue. Relaxin has beenimplicated in the dilation of cardiac and blood vessels' smooth musclecells, and has also been used in the treatment of severe chronic pain,particularly pain arising from stretching, swelling, or dislocation oftissues. Relaxin has also been shown to decrease collagen formation andsecretion, increase collagenase production, influence renalvasodilation, increase vascular endothelial growth factor expression andangiogenesis, promote dilation of blood vessels, and inhibit release ofhistamine.

In one aspect of the invention there is provided a method for thetreatment of one or more of: vascular disease including coronary arterydisease, congestive heart failure, peripheral vascular disease;treatment of arterial hypertension; diseases related to uncontrolled orabnormal collagen or fibronectin formation, including without limitationfibrotic disorders (including fibrosis of lung, heart and cardiovascularsystem, kidney and genitourinary tract, gastrointestinal system,cutaneous, rheumatologic and hepatobiliary systems); scleroderma; kidneydisease associated with vascular disease, interstitial fibrosis,glomerulosclerosis, or other kidney disorders; endometrial disordersincluding infertility due to impaired implantation; delayed onset oflabour, impaired cervical ripening, and prevention of prolonged labourdue to fetal dystocia; placental insufficiency; which comprisesadministering to a subject in need of any such treatments atherapeutically effective amount of human relaxin analog thereof asherein defined, optionally in association with one or morepharmaceutically acceptable carriers and/diluents and/or excipients.

Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Relaxin as used herein generally refers to human relaxin RLN2 unless aspecies or variant is otherwise specified, including full length relaxinor a portion of the relaxin molecule that retains biological activity.Relaxin is a peptide hormone produced by the corpora lutea of ovariesduring pregnancy in many mammalian species, including man. The secretionof the hormone into the blood stream just before parturition results ina marked softening and lengthening of the pubic symphysis and asoftening of the cervix, which facilitates the birth process. Byinhibiting uterine contractions, relaxin may influence the timing ofparturition. Like insulin, relaxin consists of two peptide chains, A andB, covalently linked by disulfide bonds. By further analogy to insulin,the two peptides are synthesized as a single-chain precursor polypeptidewith the B chain at the NH2-terminus.

In the human there are three non-allelic relaxin genes, RLN1, RLN2 andRLN3. RLN1 and RLN2 share high sequence homology. The RLN2 has twoalternatively spliced variant transcripts, isoform 1 is described inGenbank accession number NM_134441; and isoform 2, which includes analternate exon, that causes a frameshift and use of an early stop codon,is described at Genbank accession number NP_005050.2. For the purposesof the present invention, the polypeptide encoded by the isoform 1 is ofinterest.

The activities of relaxin, which are also mediated by the analogsprovided herein, are reviewed by Samuel and Hewitson (2006) KidneyInternational 69, 1498-1502; Goyce et al. (2009) Endocrinology 150:2692-2699; Westhuizen et al. (2008) Drug Discovery Today 13:640; andSamuel et al. (2007), chapter 7 in Relaxin and Related Peptides; ed.Agoulnik, Landes Bioscience; each herein specifically incorporated byreference.

The unprocessed human RLN2 polypeptide has the amino acid sequence (SEQID NO:1), where the residues comprising the mature polypeptides areunderlined:

MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTKRSLARFC

The sequence of the wild-type human mature B chain (SEQ ID NO:2) is:SWMEEVIKLCGRELVRAQIAICGMSTWS (SEQ ID NO:2). In addition to thewild-type, or naturally occurring B chain, biologically active analogsare known in the art and may be combined with the A chain analogs of thepresent invention, for example as described by Silvertown et al. (2007)FASEB J. 21, 754-765; U.S. Pat. No. 5,811,395, entitled “Relaxin analogsand derivatives methods and uses thereof”; U.S. Pat. No. 6,200,953,entitled “Relaxin analogs and derivatives compositions”; Hossain et al.(2009) Chem Biol Drug Des. 2009 January; 73(1):46-52; and Bullesbach etal. (1992) J. Biol. Chem. 267, 22957-22960; each herein specificallyincorporated by reference.

The sequence of the wild-type human RLN2 A chain isQLYSALANKCCHVGCTKRSLARFC (SEQ ID NO:3), which sequence is the referencefor describing analog sequences.

Relaxin Receptor.

Human RLN2 activates two receptors, LGR7 (RFXR1) and LGR8 (RFXR2). Asdescribed by Hsu et al. (2002) Science 295: 671-674, RLN2 activates bothof these receptors, resulting in dose-dependent increase in cAMPproduction. For reference purposes, the genetic sequences of theseproteins are known and publicly available, for example at Genbankaccession number NP_570718 (LGR8); and Genbank accession numberNM_021634 (LGR7). The selectivity and activity of RLN2 and RLN2 analogsmay be determined by various assays, including those described in theExamples herein. For example, dose dependent cyclic AMP production incells expressing one or both of the receptors may be determined;specific binding to the receptor; and assays directed at knownbiological effects of relaxin, such as the effects on connectivetissues; parturition; and the like.

Relaxin A Chain Analog.

As used herein, A chain analogs provide for one or more amino acidchanges relative to SEQ ID NO:3; and provide for altered biologicalactivity. The altered activity may be increased selectivity foractivation of the receptor LGR7; decreased selectivity for theactivation of LGR8; or increased overall receptor activation. It will beunderstood by one of skill in the art that amino acid substitutions setforth with respect to the human RLN2 A chain may also be made in the Achain of RLN2 from other animals, particularly other mammals, andparticularly placental mammals, for example as shown in the alignment ofFIG. 2. The amino acid substitutions may also be combined with otheramino acid substitutions that enhance, or that do not adversely affectthe biological activity, for example as described by Bullesbach andSchwabe (1987) J. Biol. Chem. 262, 12496-12501; Bullesbach and Schwabe(2006) J. Biol. Chem. 281, 26136-26143; and Rosengren et al. (2006) J.Biol. Chem. 281, 28287-28295, each herein specifically incorporated byreference.

A type I analog of the invention comprises an amino acid substitution atresidue 23 of a relaxin A chain, particularly a relaxin 2 A chain, forexample as set forth in Table 2. The naturally occurring amino acid maybe replaced with a small amino acid, or a conservative variationthereof. Specific substitutions of interest include Ile, Leu, Ser, Gly,Ala, and Thr. A substitution at residue 22 may also be made and combinedwith the residue 23 substitution. In some embodiments the relaxin analogcomprises the sequence:

(SEQ ID NO: 5) QLYSALANKCCHVGCTKRSLARXCwhere X is an amino acid other than phenylalanine. In some embodiments Xis selected from ala, leu, gly, ser, ile, and thr.

A type II analog of the invention comprises an amino acid substitutionat one or both of residue 16, and residue 17 of a relaxin A chain,particularly a relaxin 2 A chain, for example as set forth in Table 2.The naturally occurring amino acid may be replaced with a small aminoacid, or a conservative variation thereof. Specific substitutions ofinterest include Ile, Leu, Ser, Gly and Ala. In some embodiments therelaxin analog comprises the sequence:

(SEQ ID NO: 6) QLYSALANKCCHVGCX₁X₂RSLARFCwherein X₁ and X₂ are independently selected from amino acids other thanthreonine and lysine. In some embodiments one or both of X₁ and X₂ areIle, Leu, Gly or Ala.

Relaxin C Chain.

The wild-type, or naturally occurring RLN2 comprises a C chain, which iscleaved during maturation and is not present in the mature biologicallyactive polypeptide. The wild-type C chain of human RLN2 has thesequence: (SEQ ID NO:4)KRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKR. This sequence is fused tothe B and A chains, for example as shown in SEQ ID NO:1. In someembodiments of the invention, the C chain of RLN2 is replaced withtruncated C domain sequence, where the truncated domain consists of 2,3, 4, 5, 6, 7, 8, 9, 10 or more amino acids of the C domain, generallythe truncated domain retains the sequence at the carboxy terminus, e.g.SEQ ID NO:4, residues 1-5; residues 1-6, residues 1-7, residues 1-8; andthe like. Usually the truncated C chain will comprise the cleavagemotif, “RXXR” or “KR”. The truncated C domain may be combined wild-typeA and B chain sequences, or with analog sequences as described herein.

Polypeptides

Relaxin analogs are provided. The peptides may be provided aspharmaceutically acceptable compositions for human or animaladministration, by various therapeutic routes. Peptides are usuallyisolated in purified or homogenous form free of contaminating peptidesand proteins, or in a form of about 90-99% purity.

The relaxin analogs of the invention have altered receptor specificityas compared to the reference, naturally occurring forms. In someembodiments, a Type I analog is provided, which has a reduced affinityfor the receptor LGR8 (RFXR2); and/or an increased affinity for thereceptor LGR7 (RFXR1). In other embodiments, a Type II analog isprovided, which exhibits an enhanced bioactivity upon both receptors.Polypeptides of interest include processed forms of the relaxin analogsof the invention, which comprise an A chain and a B chain, which A chainand B chain may be linked through a disulfide bond; and unprocessedforms in which the A chain and B chain are linked through the C chain orthrough a truncated C chain. In other embodiments, an A chain analog isprovided. The A chain analogs of the invention may be combined with awild-type B chain, or with an analog thereof.

The sequence of the polypeptide may be altered in various ways known inthe art to generate targeted changes in sequence. The polypeptide willusually be substantially similar to the sequences provided herein, i.e.will differ by at least one amino acid, and may differ by at least twobut not more than about ten amino acids. The sequence changes may besubstitutions, insertions or deletions. Conservative amino acidsubstitutions typically include substitutions within the followinggroups: (glycine, alanine); (valine, isoleucine, leucine); (asparticacid, glutamic acid); (asparagine, glutamine); (serine, threonine);(lysine, arginine); or (phenylalanine, tyrosine).

Modifications of interest that do not alter primary sequence includechemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g.those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps; e.g.by exposing the polypeptide to enzymes which affect glycosylation, suchas mammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

Also included in the subject invention are polypeptides that have beenmodified using ordinary molecular biological techniques and syntheticchemistry so as to improve their resistance to proteolytic degradationor to optimize solubility properties or to render them more suitable asa therapeutic agent. For examples, the backbone of the peptide may becyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem.275:23783-23789). Analogs of such polypeptides include those containingresidues other than naturally occurring L-amino acids, e.g. D-aminoacids or non-naturally occurring synthetic amino acids.

The polypeptides of the invention biologically active molecule may beconjugated to a pharmaceutically acceptable polymer to increase itsserum half-life. The polymer may or may not have its own biologicalactivity. The suitable polymers include, for example, polyethyleneglycol (PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids,divinylether maleic anhydride, N-(2-Hydroxypropyl)-methacrylamide,dextran, dextran derivatives including dextran sulfate, polypropyleneglycol, polyoxyethylated polyol, heparin, heparin fragments,polysaccharides, cellulose and cellulose derivatives, includingmethylcellulose and carboxymethyl cellulose, starch and starchderivatives, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and α,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixturesthereof.

Polypeptides may be PEGylated, by which is meant the covalent attachmentof at least one molecule of polyethylene glycol. The average molecularweight of the reactant PEG is preferably between about 5,000 and about50,000 daltons, more preferably between about 10,000 and about 40,000daltons, and most preferably between about 15,000 and about 30,000daltons. Particularly preferred are PEGs having nominal average sizes ofabout 20,000 and about 25,000 daltons. The method of attachment is notcritical, but preferably does not alter, or only minimally alters, theactivity of the polypeptide. Preferably the increase in half-life isgreater than any decrease in biological activity. A preferred method ofattachment is via N-terminal linkage to a polypeptide.

By “increase in serum half-life” is meant the positive change incirculating half-life of a modified biologically active moleculerelative to its non-modified form. Serum half-life is measured by takingblood samples at various time points after administration of thebiologically active molecule, and determining the concentration of thatmolecule in each sample. Correlation of the serum concentration withtime allows calculation of the serum half-life. The increase isdesirably at least about two-fold, but a smaller increase may be useful,for example where it enables a satisfactory dosing regimen or avoids atoxic effect. Preferably the increase is at least about three-fold, morepreferably at least about five-fold, and most preferably at least aboutten-fold, and most preferably at least about fifteen-fold.

The polypeptide can be produced by any suitable means, such asexpression in a recombinant host cell or by chemical synthesis. Variouscommercial synthetic apparatuses are available, for example automatedsynthesizers by Applied Biosystems Inc., Foster City, Calif., Beckman,etc. By using synthesizers, naturally occurring amino acids may besubstituted with unnatural amino acids. The particular sequence and themanner of preparation will be determined by convenience, economics,purity required, and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of synthesis. A lysate may be prepared of theexpression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

The polypeptide can be linked to a polymer through any availablefunctionality using standard methods well known in the art. It ispreferred that the biologically active molecule be linked at only oneposition in order to minimize any disruption of its activity and toproduce a pharmacologically uniform product. Nonlimiting examples offunctional groups on either the polymer or biologically active moleculewhich can be used to form such linkages include amine and carboxygroups, thiol groups such as in cysteine resides, aldehydes and ketones,and hydroxy groups as can be found in serine, threonine, tyrosine,hydroxyproline and hydroxylysine residues.

The polymer can be activated by coupling a reactive group such astrichloro-s-triazine (Abuchowski et al., (1977), J. Biol. Chem.252:3582-3586), carbonylimidazole (Beauchamp et al., (1983), Anal.Biochem. 131:25-33), or succinimidyl succinate (Abuchowski et al.,(1984), Cancer Biochem. Biophys. 7:175-186) in order to react with anamine functionality on the biologically active molecule. Anothercoupling method involves formation of a glyoxylyl group on one moleculeand an aminooxy, hydrazide or semicarbazide group on the other moleculeto be conjugated (Fields and Dixon, (1968), Biochem. J. 108:883-887;Gaertner et al., (1992), Bioconjugate Chem. 3:262-268; Geoghegan andStroh, (1992), Bioconjugate Chem. 3:138-146; Gaertner et al., (1994), J.Biol. Chem. 269:7224-7230). Other methods involve formation of an activeester at a free alcohol group of the first molecule to be conjugatedusing chloroformate or disuccinimidylcarbonate, which can then beconjugated to an amine group on the other molecule to be coupled(Veronese et al., (1985), Biochem. and Biotech. 11:141-152; Nitecki etal., U.S. Pat. No. 5,089,261; Nitecki, U.S. Pat. No. 5,281,698). Otherreactive groups which may be attached via free alcohol groups are setforth in Wright, published European patent application 0 539 167 A2,which also describes the use of imidates for coupling via free aminegroups.

Pharmaceutical compositions comprising a conjugate of a biologicallyactive molecule and a polymer can be prepared by mixing the conjugatewith any pharmaceutically acceptable component, such as, for example, acarrier, a medicinal agent, an adjuvant, a diluent, and the like, aswell as combinations of any two or more thereof. Suitable pharmaceuticalcarriers, medicinal agents, adjuvants, and diluents are described in“Remington's Pharmaceutical Sciences,” 18^(th) edition, by E. W. Martin(Mack Publ. Co., Easton, Pa.).

Uses

As analogs for relaxin, the polypeptides of the invention have importantroles in the physiology of pregnancy, reproductive development,biological processes relating to smooth muscle and to connective tissue;and the like. Uses include, without limitation, induction of labor;treatment of scleroderma; reduction of hypertension, treatment ofcongestive heart failure, and treatment of other disorders associatedwith collagen and fibrinogen metabolism, including fibrosis Formulationsof polypeptides of the invention find clinical use.

Polypeptides of the invention affect epithelial cells, blood vessels,stromal cells (putative fibroblasts), and smooth muscle in the cervixand vagina, e.g. by promoting the onset of labor, increasing endometrialcells, inducing synthesis of mucins, regulating pituitary prolactin,oxytocin, and vasopressin release, etc.

These molecules also have important effects on the vascular system.Polypeptides of the invention are angiogenic in the endometrial lining,and plays a role in the attachment of the embryo to the uterus. They canbe administered to increase blood flow and vasodilation of vascularbeds. Methods for the use of relaxin to increase angiogenesis aredescribed in U.S. Pat. No. 6,211,147. Relaxin and other agonists can actas a factor in protection against arteriosclerosis and ischemic orthrombotic pathologies, by inducing dilation of blood vessels' smoothmuscle cells which results in an increment of blood flow; inhibitscoagulation processes, intensifies the fibrinolysis and lowers bloodconcentration of lipids and sodium. This effect is mediated bothdirectly, and through release NO and ANP, which largely contribute tothe effect on vessel walls and blood components. See, for example, U.S.Pat. No. 5,952,296.

Polypeptides of the invention also act as an anti-fibrinolytic agent bydecreasing collagen production, increasing collagen breakdown, andreducing the production of the collagenase inhibitor, TIMP Agonists mayact directly on stromal cells to promote remodeling of the extracellularmatrix. Remodeling of connective tissue has potential for clinicalapplications, for example in the treatment of systemic sclerosis, orscleroderma, and as a cervical softening agent at term.

Polypeptides of the invention also find use in the treatment offibromyalgia, and may also include the treatment of neurologicaldisorders, for example Alzheimer's disease, Parkinson's, and/or otherconditions such as ADD.

Another use of the polypeptides of the invention is as an analgesic andpalliative for intractable pain (see U.S. Pat. No. 5,656,592). Althoughrelaxin and other agonists can be used generally as an analgesic andpalliative for pain, the conditions most amenable to its therapeuticadministration are those in which unusual stress is chronically placedon tissues because of an acquired or inherent malformation which resultsin the displacement of tissues from their natural disposition in thebody. These agents find utility, for example, in the treatment of severechronic pain, particularly pain arising from stretching, swelling, ordislocation of tissues.

Formulations

The compounds of this invention can be incorporated into a variety offormulations for therapeutic administration.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC₅₀ asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See e.g. Finglet al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p1).

Dosage amount and interval may be adjusted individually to provide serumlevels of the active moiety which are sufficient to maintain the relaxinactivity and effects.

While human dosage levels for treating many of the above-identifiedrelaxin related diseases or disorders have yet to be optimized, a dailydose is from about 0.05 to 500.0 μg/kg of body weight per day,preferably about 5.0 to 200.0 μg/kg, and most preferably about 10.0 to100.0 μg/kg. Generally it is sought to obtain a serum concentration ofthe relaxin approximating or greater than normal circulating levels ofrelaxin in pregnancy, i.e., 1.0 ng/ml, such as 1.0 to 20 ng/ml,preferably 1.0 to 20 ng/ml.

More particularly, the compounds of the present invention can beformulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration of the compounds can be achieved invarious ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration. The agents may be systemic after administration or maybe localized by the use of an implant that acts to retain the activedose at the site of implantation.

In pharmaceutical dosage forms, the compounds may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination withother pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant is placed in proximity to the targeted site, sothat the local concentration of active agent is increased relative tothe rest of the body.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Typical dosages for systemic administration range from 0.1 μg to 100milligrams per kg weight of subject per administration. A typical dosagemay be one tablet taken from two to six times daily, or one time-releasecapsule or tablet taken once a day and containing a proportionallyhigher content of active ingredient. The time-release effect may beobtained by capsule materials that dissolve at different pH values, bycapsules that release slowly by osmotic pressure, or by any other knownmeans of controlled release.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

The use of liposomes as a delivery vehicle is one method of interest.The liposomes fuse with the cells of the target site and deliver thecontents of the lumen intracellularly. The liposomes are maintained incontact with the cells for sufficient time for fusion, using variousmeans to maintain contact, such as isolation, binding agents, and thelike. In one aspect of the invention, liposomes are designed to beaerosolized for pulmonary administration. Liposomes may be prepared withpurified proteins or peptides that mediate fusion of membranes, such asSendai virus or influenza virus, etc. The lipids may be any usefulcombination of known liposome forming lipids, including cationic lipids,such as phosphatidylcholine. The remaining lipid will normally beneutral lipids, such as cholesterol, phosphatidyl serine, phosphatidylglycerol, and the like.

Relaxin Analog Nucleic Acids

The invention includes novel nucleic acids encoding the analogpolypeptides of the invention; and fragments and derivatives thereof.Nucleic acids of the invention are synthetically produced to encode theamino acid substitutions provided by the invention, and may utilize anyappropriate combination of codons, as known in the art. For example, thecodon usage may be tailored to provide efficiency in a host organism forrecombinant production of the protein.

The subject nucleic acids can be DNA or RNA, as well as fragmentsthereof, particularly fragments that encode the biologically activepolypeptide and/or are useful in the methods disclosed herein.

The nucleic acid compositions of the subject invention can encode all ora part of the subject polypeptides. Double or single stranded fragmentscan be obtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. Isolated nucleic acids andnucleic acid fragments of the invention comprise at least about 18,about 50, about 100, to about 500 contiguous nt selected from the codingsequence. For the most part, fragments will be of at least 18 nt,usually at least 25 nt, and up to at least about 50 contiguous nt inlength or more.

The nucleic acids of the subject invention are isolated and obtained insubstantial purity, generally as other than an intact chromosome.Usually, the nucleic acids, either as DNA or RNA, will be obtainedsubstantially free of other naturally-occurring nucleic acid sequences,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant,” e.g., flanked by one or more nucleotideswith which it is not normally associated on a naturally occurringchromosome.

The nucleic acids of the invention can be provided as a linear moleculeor within a circular molecule, and can be provided within autonomouslyreplicating molecules (vectors) or within molecules without replicationsequences. Expression of the nucleic acids can be regulated by their ownor by other regulatory sequences known in the art. The nucleic acids ofthe invention can be introduced into suitable host cells using a varietyof techniques available in the art, such as transferrinpolycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Example 1 Regulation of Receptor Signaling by Relaxin a Chain MotifsDerivation of Pan-Specific and LGR7—Specific Human Relaxin Analogs

Relaxin peptides are important hormones for the regulation ofreproductive tissue remodeling and the renal cardiovascular systemduring pregnancy. Recent studies demonstrated that two of the sevenhuman relaxin family peptides, relaxin H2 (RLN2) and INSL3, signalexclusively through leucine-rich repeat-containing G protein-coupledreceptors, LGR7 and LGR8. Although it was well characterized that anRXXXRXXI motif at the RLN2 B chain confers receptor activation activity,it was previously not clear what roles RLN2 A chain plays in receptorinteraction.

Analyses of relaxin family genes on syntenic regions of model tetrapodsshowed that the A chain of RLN2 orthologs exhibited a greater sequencedivergence as compared with the receptor binding domain-containing Bchain, foreshadowing a potential role in receptor interactions; hence,defining receptor selectivity in this fast evolving peptide hormone.

To test the hypothesis that select residues in the human RLN2 A chainplay key roles in receptor interaction, we studied mutant peptides withresidue substitution(s) in the A chain. It is shown herein that alaninesubstitution at the A16 and A17 positions enhances LGR8-activationactivity of RLN2, whereas mutation at the A22-23 region (RLN2A22-23)ablates LGR8, but not LGR7, activation activity. In addition, wedemonstrated that the functional characteristics of the RLN2A22-23mutant are mainly attributed to modifications at the PheA23 position.Taken together, our studies indicated that ThrA16, LysA17, and PheA23constitute part of the receptor binding interface of human RLN2, andthat modification of these residues has led to the generation of novelhuman RLN2 analogs that would allow selective activation of human LGR7,but not LGR8, in vivo.

Earlier studies showed that the replacement of 2 arginines and 1isoleucine residue in the B chain of human RLN2 reduced the affinity bya 1000-fold, suggesting these three residues are critical for receptorinteraction. X-ray crystallographic analysis indicates that these threeresidues are concentrated and form a binding surface for interactionwith acidic residues in relaxin receptors. In addition, we have recentlyshowed that a histidine residue in the B chain of INSL3 determines thereceptor specificity of this peptide. Unlike the better characterized Bchain, studies of porcine relaxin and human INSL3 variants withsubstitutions at the N terminus of the chain A indicated that no singleamino acid in that region is functionally important.

Based on the observation that relaxin peptides from mammals exhibitdistinct receptor selectivity and that relaxin homologs underwentpositive selection in mammals, we hypothesized that structural motifstolerant of divergence in RLN2 (e.g. the C terminus of A chain) couldharbor microdomains important for the regulation of receptorselectivity; hence, the receptor activation. Consistent with ourhypothesis, studies of recombinant RLN2 peptides with point mutation(s)show that ThrA16, LysA17, and PheA23 residues in the RLN2 A chain playcritical roles in interacting with LGR7 and LGR8. In addition, theseanalyses have led to the generation of novel human RLN2 analogs thatexhibit a greater preference for LGR7 as compared with the wild typepeptide, as well as an analog that exhibits an enhanced bioactivity uponLGR8 activation.

Experimental Procedures

Syntenic Mapping and Identification of Orthologous Relaxin Genes inTetrapods.

We analyzed the genomic DNA of human (Homo sapiens), chimpanzee (Pantroglodytes), the Rhesus monkey (Macaca mulatta), rat (Rattusnorvegicus), mouse (Mus musculus), rabbit (Oryctolagus cuniculus), dog(Canis familiaris), cow (Bos taurus), pig (Sus scrofa), nine-bandedarmadillo (Dasypus novemcinctus), elephant (Loxodonta africana),Madagascar hedgehog (Echinops telfairi), gray shorttailed opossum(Monodelphis domestica), platypus (Ornithorhynchus anatinus), chicken(Gallus gallus), and the clawed frog (Xenopus tropicalis), andidentified homologs of human relaxin family genes including RLN1, RLN2,RLN3, INSL3, INSL4, INSL5, and INSL6.

The identity of relaxin family genes from different species wasdetermined by syntenic mapping and a series of reciprocal pairwisesequence comparisons using the BLAST server. Chromosome fragmentscontaining relaxin family genes syntenic to the RLN1/RLN2/INSL4/INSL6locus on the human chromosome 9 from different tetrapod species wereobtained from NCBI and Ensembl BioMart data bases based on syntenicmapping. The human-other mammal syntenic maps were downloaded from theEnsembl BioMart data mining tool. The exact locations for relaxin familygenes in the syntenic chromosome regions were also verified by BLATsearches using the UCSC Genome Bioinformatics webserver.

Subcloning of Wild Type Human Relaxin Family Genes and Variants.

Full-length cDNA for human RLN2, INSL3, and RLN3 were subcloned from ahuman testis and an ovary Marathon-ready cDNA library (Clontech,Mountain View, Calif.). To allow efficient secretion of recombinantpeptides into the conditioned media by transfected cells, cDNAs encodingthe proprotein region were appended with a signal peptide for secretionsequence from the prolactin precursor at the N terminus, and subclonedinto the mammalian expression vector, pcDNA3.1 Zeo. Expressionconstructs for mutants with point mutation(s) and/or a truncated Cdomain were generated using overlapping PCR with specific primers inthree steps. In the first PCR, forward and reverse primers were used toamplify acDNA stretch encoding the signal peptide and the downstreamsite of the introduced mutation. In the second PCR, a set of primerswere used to amplify the cDNA encoding the mutation point as well as theremaining C-terminal region of propeptide. Products from the first andthe second reactions were designed to share a 20-30-bp overlap in theregion of the introduced mutation. After purification, the mixedtemplates from the first and second PCRs were amplified with primers ofthe 5′- and 3′-ends of the propeptide coding region. The full-lengthmutant cDNAs were gel purified and ligated into the pcDNA3.1 Zeo vector.The Escherichia coli strain, Top10, was electroporated fortransformation, and the integrity of the construct was confirmed byautomated dye-terminator cycle DNA sequencing.

Expression of Relaxin Family Peptides and Variants.

For efficient detection and purification of recombinant peptides, allexpression constructs were tagged with a Myc epitope at the N terminusand a 6-histidine (His₆) tag at the C terminus of the B chain. To allowefficient cleavage of the prepropeptide during post-translationalmodifications and the secretion of mature peptides, HEK293T cells wereroutinely cotransfected with the select expression construct and aone-tenth aliquot of a convertase expression vector. Cells were culturedin Dulbecco's modified Eagle's medium/F-12 media supplemented with 10%fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin ina water-saturated atmosphere containing 5% CO₂ at 37° C. Transfectionwas performed using the calcium-phosphate precipitation method. Fourdays after transfection, conditioned media were collected, centrifuged,and filtered to remove cell debris. Recombinant peptides were thenpurified using nickel affinity chromatography.

Nickel Affinity Chromatography.

After cleared of cell debris, the conditioned medium was incubated withnickel-conjugated chelating Sepharose Fast flow resin (AmershamBiosciences) overnight at 4° C. with stirring. The resins bound withtagged peptides were washed with 10 column volumes of washing buffer(Tris-buffered saline (TBS), pH 7.5) containing 20 mM imidazole,followed by 20 column volumes of the washing buffer containing 40 mMimidazole. The bound peptides were eluted and fractionated with TBScontaining 200 mM imidazole. Fractions with immunoreactive peptides werecombined and concentrated with Amicon Ultra columns (Millipore,Billerica, Mass.; Mr cutoff, 5,000) after washing with 20 column volumesof phosphate-buffered saline. The integrity and purity of the peptideswere verified by Western blotting analysis using a monoclonal anti-Mycor an anti-relaxin B chain polyclonal antibody (Calbiochem, San Diego,Calif.) as well as Coomassie Blue staining. Purified peptides werequantified based on Western blotting analysis or a histidine peptideenzyme-linked immunosorbent assay. To ensure the fidelity of mutantpeptides, for each construct at least two different batches of peptidesfrom one half liter of conditioned medium were generated andcharacterized independently.

SDS-PAGE and Western Blotting Analysis.

Cell extracts, conditioned media, and affinity column-purified peptideswere electrophoresed using 18% SDS-PAGE before being electrotransferredto polyvinylidine difluoride membranes (Hybond-P, Amersham Biosciences)in a Trans-Blot SD semi-dry transfer cell (Bio-Rad Laboratories).Samples were mixed with a loading buffer under nonreducing or reducingconditions (100 mM dithiothreitol and 5% β2-mercaptoethanol) beforeSDS-PAGE. Blots were then washed and blocked with 5% skim milk beforeimmunoblotting using a primary antibody and a horseradishperoxidase-conjugated secondary IgG (1:5,000 final dilution in 3% skimmilk). Signals were detected following immunofluorescent imaging usingthe ECL system (Amersham Biosciences).

Histidine Peptide Enzyme-Linked Immunosorbent Assay.

To quantify purified RLN2 peptides with different mutations, serialdilutions of peptides were bound to a His tag antibody plate (Novagen,Madison, Wis.) in 1×TBS with 3% bovine serum albumin. After a 3-hincubation, the plates were washed four times with 1×TBS containing 0.1%Tween 20, followed by incubation with a rabbit anti-relaxin antibody(1:2500 in TBS with 3% bovine serum albumin). After washing andincubating with a secondary horseradish peroxidase-conjugated goatantirabbit antibody (Amersham Biosciences), the signals were detectedusing TMB luciferase substrate (Calbiochem) with a luminometer(Bio-Rad).

Receptor-Activation Analysis.

The bioactivity of relaxin family peptides was determined based on thestimulation of adenylate cyclase activity in HEK293T cells stablyexpressing recombinant LGR7 or LGR8. Stable LGR7- or LGR8-expressingcells were maintained in Dulbecco's modified Eagle's medium/F-12 mediasupplemented with 200 ng/ml Zeocin (Invitrogen). After seeding at adensity of 2×10⁵ cells per well in 500 μl of Dulbecco's modified Eagle'smedium/F-12 medium containing 0.1% bovine serum albumin in 48-wellplates, cells were preincubated at 37° C. for 30 min in the presence of0.25 mM 3-isobutyl-1-methylxanthine before treatment with increasingconcentrations of peptides (from 0 to 100 nM) in quadruplicate. Twelvehours after treatment, the culture was reacted with aceticanhydride/triethylamine, and total cAMP content in cell lysates wasmeasured using a specific cAMP radioimmunoassay. A nontagged human RLN2peptide was used as a standard to verify the bioactivity of tagged RLN2peptides.

Receptor-Binding Assay.

To determine the binding characteristics of wild type and mutantpeptides, HEK293T cells expressing LGR7 or LGR8 were grown to 90%confluence, collected by centrifugation at 3000×g for 2 min, and washedtwo times in 2 ml of ice-cold binding buffer. Cells were resuspended inice-cold binding buffer, and aliquots of cell suspension were incubatedwith increasing doses of purified recombinant peptides in the presenceof 0.025 pmol of ¹²⁵I-labeled RLN2 tracer (Phoenix Pharmaceuticals,Burlingame, Calif.) at room temperature for 2 h. After washing threetimes in an ice-cold binding buffer, radioactivity bound to the cellswas measured using a γ-counter (EG&G Wallace, Gaithersburg, Md.). Totalbinding was determined in the absence, and nonspecific binding in thepresence, of 25 nmol of unlabeled RLN2. In regular assays, total bindingwas ˜30,000 cpm, and nonspecific binding amounted to less than 20% ofthe total binding. Data points were collected in triplicate, and similarresults were obtained in at least two independent assays.

Mass Spectrometry Analysis of Recombinant Peptides.

To confirm that the recombinant relaxin family peptides were processedsimilar to that of the native peptides and assumed a nativeconformation, the mass of purified peptides was determined bymatrix-assisted laser desorption/ionization (MALDI) mass spectrometry.MALDI mass spectrometry was performed by the Pan facility at theStanford University School of Medicine. Mass spectra were acquired on aVoyager DE-RP Model (Applied Biosystems, Foster City, Calif.).

Comparative Structure Modeling.

To detect potential alterations in the surface motifs of RLN2 as aresult of mutagenesis, we generated three-dimensional models of mutantpeptides based on the human RLN2 crystal structure (Protein Data Bank6RLN) using the SWISS-MODEL server and refined by energy minimization.Molecular views were drawn with the Swiss-PdbViewer and Protein Explorer2.4 programs.

Statistical Analyses.

Receptor-activation and receptor binding activity curves generated innonlinear regression analyses (Prism; GraphPad Software, San Diego,Calif.) were evaluated relative to the samples without hormonetreatment. In all cases data are reported as the mean±S.E. of assays intriplicate or quadruplicate. Statistically significant responses(p<0.05) were determined for each stimulated response to the averagenonspecific response from control samples using analysis of variance andStudent's t test.

Results

Identification of Relaxin Homologs Syntenic to Human RLN2 inPrototherian, Metatherian, and Eutherian Mammals.

To better understand the functional motifs important for receptorinteraction in human RLN2, we analyzed the sequence conservation oforthologous relaxin peptides from mammals including monotremes,marsupials, and placentals as well as nonmammalian tetrapods. Althoughearlier studies have identified putative relaxin orthologs from a numberof vertebrates, whether some of those genes are orthologous to humanRLN2 has not been fully clarified as a result of divergent evolution ofthis family of genes in mammals. Our recent comparative genome analysesshowed that relaxin family genes evolved from three independent lociincluding, 1) INSL5 or relaxin family locusA (RFLA) corresponding tohuman chromosome 1p31; 2) RLN1/RLN2/INSL4/INSL6 or relaxin family locusB (RFLB) corresponding to human chromosome 9p24; and 3) RLN3/INSL3 orrelaxin family locus C (RFLC) corresponding to human chromosome 19p13 inthe most recent common ancestor of vertebrates. The chromosomal regionssyntenic to the human RFLB (RLN1/RLN2/INSL4/INSL6) locus could beidentified in all studied species. In all therian mammals, this locus ismarked by invariant flanking genes including RFX3, GLIS3, SLC1A1, RCL1,JAK2, C9orf46, CD274, and GLDC in neighboring loci (FIG. 1). Likewise,the single relaxin homolog at the syntenic regions of monotreme platypusand X. tropicalis is flanked by orthologous RCL1, SLC1A1, and C9orf46genes. Similar to humans, the chimpanzee genome encodes one of each ofthe orthologous RLN1, RLN2, INSL4, and INSL6 genes that cluster within a171-kb region in tandem. Unlike hominids that share a common ancestor5-10 million years ago, the Rhesus monkey genome encodes only one ofeach of the RLN, INSL4, and INSL6. On the other hand, placental mammalsincluding the dog, rat, and mouse each contain one RLN and one INSL6,whereas the cow contains only an INSL6 ortholog (FIG. 1). Unlike theplacental mammals, opossum, platypus, chicken, and X. tropicalis eachcontain only a single relaxin family gene at the syntenic RFLB locus.

These data indicate that the four human relaxin family paralogs in thislocus were likely derived from nonallelic homologous recombinations atthree separate geological times during the evolution of eutherians. Themost likely scenario is that RLN1 and RLN2 evolved before the emergenceof hominids. In contrast, INSL4 evolved before the divergence of oldworld monkeys and apes ˜15 million years ago, whereas the ancestral RLNand INSL6 genes evolved only after the emergence of eutherian mammals˜90 million years ago. Sequence comparison of RLN homologs found at thesyntenic RFLB locus of 15 mammals and that of chickens and X. tropicalisshowed that the relaxin A chain exhibits a greater sequence divergenceas compared with the RXXXRXXI motif-containing B chain (FIG. 2).

Based on this observation and earlier studies showing that relaxinorthologs from rodents exhibit a more restricted preference upon theactivation of LGR7 versus LGR8 as compared with human and pig relaxins,we reasoned that the diverging C terminus of A chain could containstructural motifs important for interacting with LGR7 and/or LGR8,hence, receptor selectivity.

Generation of Relaxin Family Peptides Using the Recombinant Approach.

To test the hypothesis that human RLN2 A chain is important for receptorinteraction, we generated and analyzed mutant RLN2 peptides with pointmutations at various positions of the C terminus of A chain. Althoughrelaxin peptides were mainly generated by synthetic chemistry approachesin earlier studies, recent studies showed that recombinant techniquesprovide an alternative approach to generate functional relaxin familypeptides and different analogs. To allow routine generation of a varietyof RLN2 mutants, we first analyzed the production of recombinantpeptides with expression constructs in which the open reading frame ofproRLN2 is tagged with a His₆ epitope at four different positions of themature peptide in addition to an N-terminal Myc epitope (WT RLN2 His1,His2, His3, and His4; FIG. 3A).

Western blotting analysis shows that peptides tagged with a His₆ epitopeat the N or C terminus of the B chain were efficiently secreted into theconditioned media, and the majority of secreted peptides exhibited apredicted 5-kDa molecular mass under reducing conditions (FIG. 3B).Similar to the nontagged wild type RLN2 peptide, RLN2 His1 and RLN2 His2peptides stimulated adenylate cyclase activity in LGR7-expressingHEK293T cells with similar potencies (FIG. 3C and Table 1).

TABLE 1 Potencies of recombinant wild type (WT) and mutant RLN2 peptideson the activation of LGR7 and LGR8 pEC₅₀ Ligand LGR7 LGR8 Nontagged RLN26.53 ± 0.10 WT RLN2 (His1) 8.66 ± 0.09 WT RLN2 (His2) 8.77 ± 0.10 RLN2C-104 8.74 ± 0.05 RLN2 C-38 8.72 ± 0.10 RLN2 C-28 8.66 ± 0.07 RLN2 C-188.74 ± 0.09 RLN2 C-8 8.81 ± 0.10 WT RLN2 9.30 ± 0.04 7.41 ± 0.14 WT RLN37.73 ± 0.07 ND^(a) WT INSL3 ND 9.03 ± 0.05 RLN2 R^(B12)A ND ND RLN2R^(B16)A ND ND RLN2 T^(A16)A 9.75 ± 0.08 8.04 ± 0.06 RLN2 K^(A17)A 9.18± 0.07 8.39 ± 0.06 RLN2 R^(A16)A 9.67 ± 0.10 7.56 ± 0.04 RLN2^(A19-20)8.56 ± 0.07 8.41 ± 0.07 RLN2^(A22-23) 8.87 ± 0.07 ND RLN2 R^(A22)A 10.03± 0.07  7.51 ± 0.09 RLN2 P^(A23)A 9.45 ± 0.09 ND RLN2 K^(A17)G 9.04 ±0.05 8.28 ± 0.05 RLN2 K^(A17)Q 9.31 ± 0.11 7.77 ± 0.05 RLN2 K^(A17)D8.41 ± 0.06 8.36 ± 0.06 ^(a)ND, the pEC₅₀ values were not measurable.

Based on these observations, we tagged all expression constructs with anN terminus Myc epitope and a C terminus His₆ epitope flanking the matureB chain in subsequent studies. In addition, given that relaxin familypeptides contain cryptic C domains of varying lengths and with multiplebasic residues that could be subjected to alternative post-translationalprocessing, we explored the possibility of generating relaxin peptideswith a uniform C domain linker sequence to avoid differential processingof the secreted peptides. Studies of four expression constructs, inwhich the B and A chains of RLN2 were linked with a truncated C-domainof 8, 18, 28, or 38 amino acids (FIG. 4A, RLN2 C-8, RLN2 C-18, RLN2C-28, and RLN2 C-38), revealed that the expression construct with as fewas an 8-amino acid linker sequence allows efficient production of RLN2in conditioned media (FIGS. 4B and 5B).

Functional analyses showed that RLN2 generated using the RLN2 C-8, RLN2C-18, RLN2 C-28, or RLN2 C-38 constructs all have an EC₅₀ upon LGR7activation similar to that of RLN2 derived from the construct containinga 104-amino acid C domain sequence (FIG. 4C and Table 1). Similarly,receptor-binding analysis showed that the Myc epitope-tagged RLN2,derived from the RLN2 C-8 construct, competed for labeled nontagged RLN2binding to LGR7 with a high affinity (FIG. 4D and Table 2).Subsequently, all our mutant expression constructs were engineered withan 8-amino acid miniature linker sequence between the B and A chains.Using the same approach, we also generated recombinant INSL3 and RLN3.Mass spectrometry analysis of purified peptides confirmed that theserecombinant peptides are processed as heterodimeric peptides afterpost-translational cleavage of the C domain sequences (Table 3).

Importantly, functional analysis showed that recombinant peptidesexhibit receptor activation characteristics similar to that of purifiedor chemically synthesized peptides. Unlike RLN3 and INSL3, whichselectively activated LGR7 and LGR8, respectively, RLN2 activates bothreceptors at the nanomolar range (FIG. 5A). Furthermore, we show thatalanine substitution at either of the charged residues of the RXXXRXXImotif (ArgB12 and ArgB16) ablated both the LGR7 and the LGR8 activationactivity of RLN2 (FIG. 5A).

A16-17 and A22-23 Regions in the RLN2 A Chain Play Critical Roles inReceptor Interaction.

To investigate the role of residues at the C terminus of A chain inreceptor interaction, we generated five mutants with alaninesubstitution at five different positions (FIG. 6A, RLN2 TA16A, RLN2KA17A, RLN2 RA18A, RLN2A19-20, and RLN2A22-23). Western blottinganalysis showed that all five mutants were processed to the mature form(FIG. 5B). Whereas RLN2 TA16A, RLN2 KA17A, and RLN2 RA18A mutantsexhibited an EC₅₀ on LGR7 activation similar to that of the wild typepeptides, RLN2A19-20 and RLN2A22-23 mutants exhibited decreasedLGR7-activation activities (FIG. 6B, left panel, and Table 1). Inaddition, receptor-binding analyses shows that, except for RLN2A22-23,which exhibited a reduced affinity for LGR7, all other mutants competedfor labeled RLN2 binding to LGR7 with affinities similar to that of wildtype peptide (FIG. 6C, left panel, Table 2).

Although alanine substitution has minimal effects on maximumLGR7-activation activity of mutant peptides, mutation at the RLN2A16 andRLN2A17 positions leads to a 2-fold increase of the maximumLGR8-activation activity (FIG. 6B, right panel). In contrast, mutationof the RLN2A22-23 region almost abolished the LGR8-activation activityof RLN2. Consistent with analyses of receptor-activation activity, theRLN2 TA16A and RLN2 KA17A mutants exhibited an IC₅₀ value similar tothat of wild type RLN2 for both LGR7 and LGR8, whereas the RLN2A22-23mutant lost its LGR8-binding activity (FIG. 6C and Table 2).

The Functional Characteristics of RLN2A22-23 Mutant are Attributed toMutation at the PheA23 Position.

Given the findings that the A22-23 region plays important roles inreceptor signaling, we then analyzed mutants with point mutation atArgA22 and PheA23 positions (FIGS. 5B and 7A). Functional analysisshowed that alanine substitution at the ArgA22 position has minimaleffect on the LGR7- or LGR8-activation activity (FIG. 7B). In contrast,the RLN2 FA23A mutant lacks an LGR8-activation activity, but retains anLGR7-activation activity similar to that of wild type peptides.Receptor-binding assays showed that the RLN2 RA22A and RLN2 FA23Amutants bind to LGR7 with affinities similar to that of wild typepeptides (FIG. 7C). In contrast, alanine substitution at the PheA23position significantly reduced the affinity for LGR8 (FIG. 7C).

ThrA16, LysA17, and PheA23 Residues Constitute Part of theReceptor-Interacting Interface in Human RLN2.

To gain a better understanding of the molecular basis for the regulationof receptor activation by ThrA16, LysA17, and PheA23, we generatedsimulated structure models of RLN2 mutants using the SWISS-MODEL server(FIG. 8). Comparative structure analyses showed that the LysA17 residue(brown filled space) is embedded between the interface of the A chain(white filled space) and the B chain (green dot space), and is the onlyA chain residue with its side chain exposed on the same surface withArgB12, ArgB16, and IleB19 of the RXXXRXXI motif (FIG. 8, left panel).Alanine substitution in the RLN2 KA17A mutant eliminated the extendedside chain of lysine that protrudes toward the B chain surface (FIG. 8,middle panel). Unlike the LysA17 residue, the ThrA16 and PheA23 residuesare located opposite to the surface including LysA17 and the RXXXRXXImotif (FIG. 8, right panel). Judging by the orientation and the distanceof these residues to the binding motif on the B chain, it likelyinteracts with a distinct ligand-binding interface of LGR7 and LGR8. Thefinding that the elimination of the extended side chain of lysine at theLysA17 position leads to an increased LGR8-activation activity was ofparticular interest considering its close proximity to the RXXXRXXImotif.

To further characterize the importance of the LysA17 position inreceptor interaction, we analyzed the receptor activation activity ofmutants with an aspartic acid, glutamine, or glycine at this position(FIG. 5B). Unlike the pan-specific RLN2 KA17A mutant, peptides with aglycine or glutamine at the LysA17 position exhibited normal LGR7- andLGR8-activation activity (FIG. 9, A and B). Alternatively, substitutionwith an aspartic acid residue at the same position led to a reducedLGR7-activation activity (FIG. 9A). Receptor-binding assays showed thatsubstitution with a glycine and aspartic acid at LysA17 reducedLGR7-binding activity by 3-fold (FIG. 9B). Conversely, glutaminesubstitution at LysA17 had a minimal effect on LGR7- or LGR8-bindingactivity.

Based on analyses of the receptor-activation and receptor bindingactivities, our study identified ThrA16, LysA17, and PheA23 of RLN2 ascrucial residues in the interaction with LGR7 and LGR8, and suggestedthat these three residues act cooperatively with the well characterizedRXXXRXXI motif in shaping the functional characteristics of human RLN2.These studies have also led to the generation of LGR7-specific humanRLN2 analogs that are useful for selective activation of LGR7 signalingpathways in patients as well as a pan-specific analog with an enhancedLGR8-activation activity.

Relaxin family peptides are structurally similar to insulin and arefirst synthesized as a single chain prorelaxin, in which a C domainconnecting the B and A chains is excised during the post-translationalmodification. Based on studies of insulin, which shares a similar threedisulfide bridged two chain architecture, the C domain is thought toplay a minimal role in the folding of a mature relaxin peptide. We showthat, similar to insulin, a minimal linker sequence with only 8 aminoacids is sufficient for the generation of mature RLN2, INSL3, and RLN3peptides in transfected cells. These results suggest that theconformational propensities for a mature two-chain relaxin familypeptide does not require a native C domain, and are consistent with theobservation that the C domain sequence of these genes diverged greatlyeven among closely related species.

Unlike the C domain sequences, earlier studies of insulin and relaxinbiosynthesis have shown that the A chain may function mainly as ascaffold in the assembly of a bioactive disulfide-bridged maturepeptide. We show that RLN2 mutants with substitutions at variousresidues of the C terminus of the A chain were processed to matureforms, suggesting these residues play a minimal role in disulfidepairing and the folding of mature peptides.

Earlier structural-functional relationships of relaxin family peptideshave been inferred from the degree of residue conservation amongmammalian species and studies of native receptors. An invariant RXXXRXXImotif in the B chain was found to be essential for receptor binding, andreplacement of arginines or isoleucine in this motif greatly reduced thebinding activity. Although the molecular basis for the interactionbetween RXXXRXXI motif and the receptor is not clear, x-raycrystallographic structure analysis shows that the conserved argininesand isoleucine form a tight receptor-binding surface (34). Based onthese observations, it was proposed that interactions between thesecharged residues and select acidic residues within the concave face ofthe ectodomain of LGR7 are critical to the receptor activation (58). Incontrast, it was hypothesized that the isoleucine residue of the RLN2 Bchain could interact with a cluster of tryptophan, isoleucine, andleucine residues close to the ligand-interacting acidic residues throughhydrophobic interactions (38, 58). Similar to relaxin, a pair ofarginines and a histidine in the INSL3 B chain were shown to be criticalfor receptor interaction (35, 37, 38, 50, 59). Interestingly, relaxinorthologs from placental mammals display varied preference for the tworelaxin receptors even though all these orthologs contain the invariantRXXXRXXI motif (8, 40). Based on this observation, we hypothesizedthat: 1) the expansion of family genes at the syntenic RFLB locus inplacental mammals has allowed the divergence in functionalcharacteristics of relaxin peptides unrelated to the core structure ofthese peptides; and 2) analysis of residues at sites tolerant ofsubstitutions could reveal molecular determinants that are critical tothe optimal signaling in a select lineage. Indeed, in contrast to thegeneral notion that the A chain has a minimal role in receptorinteraction, we show that mutations at ThrA16, LysA17, and PheA23positions significantly alter the receptor-binding andreceptoractivation activities of RLN2. Whereas PheA23 is important forhigh affinity binding to LGR8, ThrA16 and LysA17 play a more criticalrole in restricting the interaction with LGR8. These findings areconsistent with the hypothesis that residues at sites tolerant ofsubstitutions in a rapidly expanding protein family could representcritical motifs important for lineage-specific adaptations. However,whether the difference in receptor selectivity of relaxin peptides fromdifferent mammals was attributed to residue difference at the C terminusof A chain remains to be investigated. Our study effectively expandedthe known receptor-interacting interface of human RLN2, and indicatedthat ThrA16, LysA17, and PheA23 at the C terminus of the A chain,together with the RXXXRXXI motif, constitute a broad receptor bindinginterface for optimal signal transmission. Of interest, structuralmodeling analyses show that LysA17 is the only residue with its sidechain protruding onto the same surface with the RXXXRXXI motif and is inclose proximity with ArgB12, ArgB16, and IleB19. Therefore, it is likelythat LysA17 and the three receptor-binding residues in the RXXXRXXImotif form an extended binding interface to interact with the receptor.In contrast, ThrA16 and PheA23 are positioned on a surface opposite tothat of the RXXXRXXI motif, suggesting that these residues couldinteract with a receptor interface distant from that for LysA17. It isimportant to note that depending on the amino acid introduced at theLysA17 position, the preference for LGR8 could be either enhanced orreduced. Whereas alanine substitution enhances LGR8-activation activity,corresponding substitution with a polar or a reverse charged residuereduces it. Therefore, the side chain at the LysA17 position could beconductive to conformational transformation and an alanine residueprovides an induced fit for LGR8 activation. In contrast, a polar or areversed charged residue at this position increases the hindrance forthe adoption of an activation configuration for LGR8. In the last fewyears, human RLN2 has been the subject of various clinical studies aimedto treat etiology in reproduction and other systems (25-28). Based onthe understanding that: 1) RLN2 activates LGR8 at the nanomolarconcentration; and 2) LGR8 signaling is involved in the regulation ofoocyte maturation, ovarian follicle development, and the positioning ofthe ovary (10, 22, 23, 60), it is conceivable that clinical applicationof the indiscriminative wild type human RLN2 could impose unwanted sideeffects on LGR8-mediated physiological processes in patients. Thefinding that RLN2A22-23, and FA23A mutants preferentially activate LGR7thus provides novel agents to allow selective activation ofLGR7-mediated physiological processes in patients. Although the relatedRLN3 activates only LGR7, but not LGR8, it also functions as a potentagonist for GPCR135 and GPCR142. In contrast, the RLN2 TA16A and RLN2KA17A mutants, which exhibit high potencies on the activation of bothLGR7 and LGR8, function as pan-specific agonists for these tworeceptors. These reagents could be useful in therapeutic applicationsthat aim to activate both LGR7 and LGR8 in patients.

TABLE 2 Potencies of wild type (WT) and mutant RLN2 peptides incompetition for RLN2 binding to LGR7 and LGR8 pIC₅₀ Ligand LGR7 LGR8Nontagged RLN2 8.78 ± 0.12 RLN2 C-8 8.61 ± 0.09 WT RLN2 8.72 ± 0.06 7.90± 0.16 RLN2 T^(A16)A 8.40 ± 0.06 7.74 ± 0.07 RLN2 K^(A17)A 8.36 ± 0.088.14 ± 0.18 RLN2 R^(A18)A 8.42 ± 0.08 7.46 ± 0.17 RLN2^(A19-20) 8.21 ±0.10 7.51 ± 0.12 RLN2^(A22-23) 7.50 ± 0.16 ND^(a) RLN2 R^(A22)A 8.42 ±0.08 6.80 ± 0.32 RLN2 P^(A23)A 8.17 ± 0.06 ND RLN2 K^(A17)G 7.57 ± 0.227.40 ± 0.41 RLN2 K^(A17)Q 7.93 ± 0.15 7.96 ± 0.11 RLN2 K^(A17)D 7.75 ±0.25 7.53 ± 0.24 ^(a)ND, the pIC₅₀ values were not measurable.

TABLE 3 Mass spectrometry analysis of purified recombinant peptidesPeptide Molecular weight measured Expected molecular weight WT RLN2 82248225 WT RLN3 7888 7888 WT INSL3 8270 8272Abbreviations used are: RLN1, relaxin H1; RLN2, relaxin H2; RLN3,relaxin 3; INSL3, insulin-like 3; GPCR, G protein-coupled receptor;LGR7, leucine-rich repeat-containing GPCR 7; LGR8, leucine-richrepeat-containing GPCR 8; MALDI, matrix-assisted laserdesorption/ionization; TBS, Tris-buffered saline.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such a disclosure byvirtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method of treating preeclampsia, comprisingadministering to an individual an effective dose of a pharmaceuticalformulation comprising a purified polypeptide analog of a mammalianrelaxin 2 (RLN2) protein comprising an amino acid substitution at one orboth of amino acid residues 16 and 17 to an amino acid other than thewild-type in the A chain, wherein the analog has increased activity foractivation of the relaxin receptors LGR7 or LGR8 relative to the wildtype protein; and a pharmaceutically acceptable excipient.
 2. The methodof claim 1, wherein the polypeptide is a human RLN2 polypeptidecomprising a substitution at residue 16 to an amino acid other thanthreonine.
 3. The method of claim 1, wherein the polypeptide is a humanRLN2 polypeptide comprising a substitution at residue 17 to an aminoacid other than lysine.
 4. The method of claim 1, wherein the amino acidat residue 16 or 17 is selected from alanine, glycine, isoleucine andleucine.
 5. The method of claim 1, wherein the polypeptide analog iscovalently linked to a RLN2 B chain.
 6. The method of claim 5, whereinthe RLN2 B chain has a wild-type amino acid sequence.
 7. The method ofclaim 5, wherein the RLN2 B chain is a variant of the wild-type RLN2 Bchain amino acid sequence.
 8. The method of claim 1, wherein the A chainand the B chain are covalently linked to a relaxin C chain.
 9. Themethod of claim 8, wherein the C chain is a truncated C chain.
 10. Themethod of claim 1 wherein the effective dose is from 0.05 to 500 μg/kg.11. The method of claim 1, wherein the effective dose is from 5 to 200μg/kg.